View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Covenant University Repository Biotechnology Advances 29 (2011) 210–222 Contents lists available at ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv Research review paper Advances in plant molecular farming Olawole O. Obembe a,⁎, Jacob O. Popoola a, Sadhu Leelavathi b, Siva V. Reddy b a Department of Biological Sciences, Covenant University, PMB 1023 Ota, Ogun State, Nigeria b Plant Transformation Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India article info abstract Article history: Plant molecular farming (PMF) is a new branch of plant biotechnology, where plants are engineered to Received 27 July 2010 produce recombinant pharmaceutical and industrial proteins in large quantities. As an emerging subdivision Received in revised form 12 November 2010 of the biopharmaceutical industry, PMF is still trying to gain comparable social acceptance as the already Accepted 12 November 2010 established production systems that produce these high valued proteins in microbial, yeast, or mammalian Available online 27 November 2010 expression systems. This article reviews the various cost-effective technologies and strategies, which are being developed to improve yield and quality of the plant-derived pharmaceuticals, thereby making plant- Keywords: Plant-derived proteins based production system suitable alternatives to the existing systems. It also attempts to overview the Clinical development different novel plant-derived pharmaceuticals and non-pharmaceutical protein products that are at various Commercialization stages of clinical development or commercialization. It then discusses the biosafety and regulatory issues, Biosafety which are crucial (if strictly adhered to) to eliminating potential health and environmental risks, which in Regulation turn is necessary to earning favorable public perception, thus ensuring the success of the industry. © 2010 Elsevier Inc. All rights reserved. Contents 1. Introduction .............................................................. 211 2. Plant transformation strategies ..................................................... 211 2.1. Stable nuclear transformation................................................... 211 2.2. Stable plastid transformation ................................................... 211 2.3. Plant cell-suspension cultures................................................... 211 2.4. Transient expression systems ................................................... 212 2.4.1. Agroinfiltration method ................................................. 212 2.4.2. Virus infection method ................................................. 212 2.4.3. The magnifection technology............................................... 212 3. Limitations and optimization of plant production systems......................................... 212 3.1. The problem of low yield..................................................... 212 3.1.1. Optimizing transcript expression ............................................. 212 3.1.2. Optimizing protein's stability............................................... 213 3.2. The glycosylation challenge.................................................... 213 3.3. Choice of suitable host plants ................................................... 213 3.3.1. Cost of downstream processing ............................................. 214 4. Overview of plant-derived recombinant proteins ............................................. 215 4.1. Plant-derived vaccine antigens .................................................. 215 4.2. Plant-derived antibodies ..................................................... 215 4.3. Therapeutic and nutraceutical proteins .............................................. 217 4.4. Non-pharmaceutical plant-derived proteins ............................................ 217 5. Biosafety and regulatory issues ..................................................... 217 5.1. Problem of co-mingling and contamination of the food/feed chain ................................. 217 5.2. The problem of gene spread and unintended exposures ...................................... 218 5.3. Risk of horizontal gene transfer .................................................. 218 6. Concluding remarks. .......................................................... 218 ⁎ Corresponding author. Tel.: +234 8130928965. E-mail address: [email protected] (O.O. Obembe). 0734-9750/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2010.11.004 O.O. Obembe et al. / Biotechnology Advances 29 (2011) 210–222 211 Acknowledgement ............................................................. 218 References ................................................................. 218 1. Introduction recombinant proteins till date. The method has been used to accumulate protein in the dry seeds of cereals, which allows long- Plant molecular farming (PMF) refers to the production of term storage of the seed at room temperature without degradation of recombinant proteins (including pharmaceuticals and industrial the protein (Horn et al., 2004). Additionally, the system has high proteins) and other secondary metabolites, in plants. This involves scale-up capacity, as crops such as the cereals are grown practically the growing, harvesting, transport, storage, and downstream proces- everywhere. However, a long production cycle of some crops and the sing of extraction and purification of the protein (Wilde et al., 2002). potential to cross with native species or food crops are limiting public This technology hinges on the genetic transformability of plants, acceptance of this method (Commandeur et al., 2003). which was first demonstrated in the 1980s (Bevan et al., 1983). The first recombinant plant-derived pharmaceutical protein (the human 2.2. Stable plastid transformation growth hormone) and the first recombinant antibody (expressed in the progeny of the cross of two individual transgenic plants Plastid transformation provides a valuable alternative to nuclear expressing single immunoglobulin gamma and kappa chains) were transformation because it combines numerous advantages that the produced in transgenic plants in 1986 and 1989, respectively (Hiatt nuclear transformation lacks, including the provision of a natural et al., 1989; Barta et al., 1986). However, it was not until 1997 that the biocontainment of transgene flow by out-crossing (as plastids are first recombinant protein, avidin (an egg protein) was expressed in inherited through maternal tissues in most species and the pollen transgenic maize for commercial purpose (Hood et al., 1997). These does not contain chloroplasts, hence the transgene may not be exploits really demonstrated that plants could be turned into bio- transferable, thereby allaying public concerns (Cardi et al., 2010; factories for the large scale production of recombinant proteins. It has Meyers et al., 2010). Transgenic plants with homoplastomic chloro- been proven over the years that plants have the ability to produce plast transformation (in which every chloroplast carries the trans- even more complex functional mammalian proteins with therapeutic gene) are selected after several generations of plant regeneration activity, such as human serum proteins and growth regulators, from bombarded leaf explants, on selection medium containing antibodies, vaccines, hormones, cytokines, enzymes and antibodies spectinomycin or in combination with streptomycin. Our group (Liénard et al., 2007). This has been possible due to their ability to (Reddy et al., 2002; Sadhu and Reddy, 2003) had previously achieved perform post-translational modifications that make these recombi- accumulation of a bacterial- and a human therapeutic protein, up to nant proteins fold properly and maintain their structural and 3–6% of total soluble proteins in tobacco chloroplasts. Recently, Oey functional integrity. et al. (2009) reported a whopping expression level (70% of the total The fast-growing market for recombinant biopharmaceuticals, soluble proteins) for a proteinaceous antibiotic, with the chloroplast which accounted for about 10% of the pharmaceutical market in 2007 system, which represents the highest recombinant protein accumu- (Lowe and Jones, 2007), had been predicted to expand to US $ lation achieved so far in plants. This enormous potentials notwith- 100 billion in 2010 (Knäblein, 2005). With increasing demand for bio- standing, plastid transformation is still limited in its applications, in pharmaceuticals, coupled with the high costs and inefficiency of the that even though it has been achieved in other plant species such as existing production systems (Knäblein, 2005), which include yeast, tomato, eggplant, lettuce, and soybeans (Bock, 2007; Singh and microbes, animals cells (Jones et al., 2003) and transgenic animals Verma, 2010), routine chloroplast transformation is only possible in (Harvey et al., 2002), there now exists a shortage in manufacturing tobacco, which is inedible and highly regulated, being rich in toxic capacity, to such an extent that some patients have to wait for these alkaloids. It is also envisaged that protein stability will change over products. Hence transgenic plants are gaining more attention as the time even with refrigeration (Horn et al., 2004). new generation bio-reactors; with active research and development activity in PMF identified